Molecular Plant Breeding 2025, Vol.16, No.4, 241-249 http://genbreedpublisher.com/index.php/mpb 245 CmSPS1 promotes sucrose synthesis in all fruits, while sucrose synthase 2 is expressed more in respiratory leap varieties, whose hexose levels are generally lower (Dai et al., 2011; Stroka et al., 2024). Some transcription factors, such as CmMYB44 and CmERFI-2, also affect sugar accumulation by regulating the expression of key genes (such as CmSPS1 and CmACO1) (Figure 2) (Gao et al., 2023; Wang et al., 2023a). Other genes related to glucose metabolism, such as CmINH3, CmTPP1, and CmTPS5/7/9, also have special expressions at the fruit ripening stage, suggesting that they may also be involved in regulating glucose accumulation. Figure 2 CmMYB44 directly binds to the promoters of CmSPS1 and CmACO1 and acts as a transcriptional repressor (Adopted from Gao et al., 2023) Image caption: A) Y1H analysis of CmMYB44 binding to the promoters of CmSPS1 and CmACO1. The growth status of transformed yeasts on two mediums. Normal yeast growth on a defective medium containing the antibiotic Aureobasidin A indicates CmMYB44 can bind to the promoters of CmSPS1 and CmACO1. B and C) Luciferase reporter assay showed the binding of CmMYB44 to the CmSPS1 and CmACO1 promoters in vivo. The infected Nicotiana benthamiana was measured by a living fluorescence imager. The fluorescence intensity weaker than control (empty vector) means that CmMYB44interacts with promoter, and represses the gene expression. D and E) GUS activity assays analysis of CmMYB44 binding to the CmSPS1 and CmACO1 promoters in N. benthamiana leaves. Relative GUS activity decreasing indicates transcriptional regulation is repressed. An independent t-test was used to analyze the difference between the treatment group and control. Significant differences are marked with asterisks (**P < 0.01). Error bars are shown with the three biological replicates. F) Electrophoretic mobility shift assay (EMSA) analysis of CmMYB44 binding to the CmSPS1 and CmACO1 promoters. The hot probe was biotin-labeled CmSPS1 and CmACO1 promoters, while the cold probe was a nonlabeled competitive probe (with a 50/100-fold higher concentration than the hot probe). His-tagged CmMYB44 (CmMYB44-His) was purified and used for DNA-binding assays. The sequence of the biotin-labeled probe is shown and the CCGTTG motif is highlighted in bold (Adopted from Gao et al., 2023) 7.3 Potential gene targets for regulating sugar accumulation under humidity stress There are some key genes that can be targeted for improvement regarding the impact of humidity on sugar content. CmACO1, CmERFV-2, and CmERFI-5 in the ethylene pathway, CmSPS1 and CmVINV2 in sucrose metabolism, CmSWEET10 in glucose transport, and the transcription factors CmMYB44 and CmPIF8 that regulate them, are all very important targets (Zhou et al., 2023; Guan et al., 2024; Yang et al., 2025). Wen et al. (2025) indicated that proteins like CmFLA8 might also have an inhibitory effect on fruit development, and their expression would be influenced by hormones and stress signals. If these key genes can be controlled through gene editing or molecular markers, it is possible to improve the sugar accumulation capacity and fruit quality of melons under different humidity conditions (Lao et al., 2023; Ren et al., 2023). 8 Case Study: Controlled Humidity Effects on Sugar Accumulation in Greenhouse Melons 8.1 Experimental design: RH treatments in commercial greenhouses In commercial greenhouses, researchers often observe the growth of melons and changes in fruit quality by adjusting the air humidity (RH). Some experiments set normal humidity and fogging treatment (increasing humidity), and combined with different substrate moisture contents (such as 100%, 130%, 160% field capacity) to
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